Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of CD4+CD25+Foxp3+ regulatory T cells (Treg). Our recent studies have focused on defining the mechanism of action of the CD4+CD25+Foxp3+ Treg cells in vitro and on an analysis of their potential dysfunction in autoimmune disease: (1) Recent studies have defined naturally occurring Treg as expressing the forkhead family transcription factor, Foxp3. Recent reports have demonstrated that peripheral CD4+Foxp3- T cells can be induced in vitro to express Foxp3 in the presence of TGF-beta. These induced cells, termed iTregs, are functionally similar to thymic-derived Tregs in that they are anergic, suppressive, and capable of inhibiting disease in vivo. We have examined the cytokine and costimulatory molecule requirements for the TGF-beta-mediated induction and maintenance of Foxp3 by CD4+CD25- Foxp3- cells. Although signalling via CD28 is not required, IL-2 plays a critical role in the induction, but not the maintenance of Foxp3 expression in vitro and in vivo. One major question that remains unresolved by these studies is whether and under what conditions iTreg are generated in vivo. The therapeutic potential of TGF-beta iTreg also remains to be determined. Our results indicate that T cells that have been recently activated in vitro under Th1, Th2, or non-polarizing conditions cannot be induced to express Foxp3 under the conditions we have used for naive cells. It is unclear whether autoantigen-specific memory cells that might be isolated from patients will also be difficult to convert to iTreg expressing Foxp3. (2) While it is clear that the addition of exogenous TGF-beta plays a critical role in the induction of Treg, the role of TGF-beta produced by Treg remains controversial. To further analyze the contribution of TGF-beta in Treg function, we have used recombinant TGF-beta latency associated peptide (LAP) to neutralize active TGF-beta. LAP had no effect on the suppressive effects of Foxp3+ Treg from Foxp3-GFP knock in mice. In contrast, LAP modestly reversed the suppressive effects of CD4+CD25+ T cells from conventional mice suggesting that Treg- associated TGF-beta plays a role in suppressing the responses of activated T cells. Co-culture of pre-activated Foxp3+ Treg and nave Foxp3- CD4+ T cells in the presence of anti-CD3 and IL-2 results in the induction of Foxp3 expression in 10-20% of the responder cells. This conversion is dependent on TGF-beta, as it can be blocked with LAP and requires contact between the Treg and effector T cells. The activation of TGF-beta and the subsequent Foxp3 induction are not dependent on expression of the alpha-V integrin on either the Foxp3+ Treg or the responder T cells. Antigen presenting cells are also not necessary, as induction of Foxp3 expression is seen with plate-bound anti-CD3. The induced CD4+FoxP3+ T cells are suppressive when co-cultured with fresh CD4+Foxp3- T cells in vitro and when mixed with CD4+Foxp3- T cells in vivo. Thus, it appears that Treg associated TGF-beta can play a role in suppression of activated effectors, but more importantly can mediate infectious tolerance by converting conventional T cells into Treg. (3) Although expression of Foxp3 is considered the definitive marker of mouse Treg cells, several studies have suggested that this is not the case in man because T cell receptor stimulation alone has been reported to induce Foxp3 expression in nave human CD4+CD25- T cells. We have examined in depth the requirements for de novo induction of Foxp3 in non-regulatory CD4+Foxp3- T cells and the potential contribution of TGF-beta in Foxp3 induction. In contrast to previous studies, we have demonstrated that human CD4+Foxp3- T cells resemble mouse CD4+Foxp3- cells. TCR stimulation alone was insufficient to induce Foxp3 expression in the absence of TGF-beta, although high levels of Foxp3 could be induced in the presence of TGF-beta. Foxp3 expression in the TGF-beta-induced cells was stable for several weeks in culture in the presence of IL-2. In contrast, to TGF-beta-induced CD4+Foxp3+ cells that were both anergic and suppressive in vitro, human CD4+Foxp3+ cells were neither anergic nor suppressive and produced high levels of effector cytokines. The failure of human TGF-beta-induced CD4+Foxp3+ cells to exhibit regulatory activity raises the possibility that even high levels of Foxp3 expression are insufficient to define a cell as a Treg and that other factors, present in mouse CD4+ T cells, may be required to act in concert with Foxp3. These studies suggest that considerable caution should be exercised before concluding that a Foxp3+ human T cell is a bona fide Treg. As TGF-beta is found at high levels at inflammatory sites, some of the CD4+CD25+Foxp3+ cells isolated from such tissues may resemble the Foxp3+ non-Treg we have generated in vitro. (4) Recent studies have identified a new subset of effector T cells that produce IL-17 that are known as Th17 cells and play a major role in the pathophysiology of inflammatory disease and defense against extracellular bacteria and fungi. The cytokines responsible for the differentiation of Th17 cells are TGF-beta and IL-6. In conjunction with T cell receptor stimulation, these cytokines induce expression of the orphan nuclear receptor RORgammat that is critical for Th17 generation. As we have shown that TGF-beta alone promotes expression of Foxp3, while the combination of TGF-beta and IL-2 suppresses Foxp3 and enhances IL-17 production, the differentiation of Treg and Th17 appears to be reciprocally regulated. Because of our studies demonstrating the importance of IL-2 for the generation of Treg in vitro, we examined the role of IL-2 in Th17 generation and demonstrated that a previously unrecognized function of IL-2 is to constrain Th17 production. Genetic deletion or antibody blockade of IL-2 promoted Th17 generation. Whereas STAT3 was a key regulatory of RORgammat and IL-17 expression, absence of IL-2 or disruption of its signaling by deletion of STAT5 resulted in enhanced Th17 development. It is notable that IL-2, the major cytokine that promotes clonal expansion, inhibits the generation of a highly inflammatory T cell subset. Thus, one important role of IL-2 may be to limit inflammation during an immune response by facilitating the generation of Treg and blocking differentiation of Th17 cells.